Feb 23, 2026
Version 2
HEdeX protocol V.2
- 1Johns Hopkins University
- Fan Wu
Protocol Citation: Fan Wu 2026. HEdeX protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnz9wvmk/v2Version created by wu.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 21, 2026
Last Modified: February 23, 2026
Protocol Integer ID: 243763
Keywords: hedex protocol, protocol
Abstract
For destaining H&E and restaining
Decoverslipping
1d
Soak the H&E stained slides in 100% acetone
1d
Rehydration (Eosin destaining)
45m
Soak the H&E slides without coverslip in 100% Xylene
5m
Soak the H&E slides without coverslip in 100% EtOH
5m
Soak the H&E slides without coverslip in 90% EtOH
5m
Soak the H&E slides without coverslip in 80% EtOH
5m
Soak the H&E slides without coverslip in 75% EtOH
5m
Soak the H&E slides without coverslip in 100% H2O
5m
PBS wash for 3 times, 5 minutes each
15m
Destaining (Hematoxylin destaining)
35m
Prepare the destaining solution and mildly soak the slides into the destaining solution (70ml of PBS + 30 ml of H2O + 500ul of concentrated 37% HCl).
Dip the slide in destaining solution for several times and let it set for 5 minutes. Repeat this step for 3 times.
15m
Wash the slide in EDTA buffer solution (5 mM) for 5 minutes.
5m
PBS wash for 3 times, 5 minutes each
15m
Antigen retrieval
35m
Add 300 ml of unmasking solution (Antigen unmasking solution, citric acid based, vector, H-3300) into 32 ml of H2O in a small plate (heat resistant plate, maybe the cap of the antibody box), make sure the water covers the slides.
20m
TBST wash for 3 times, 5 minutes each
15m
iCLAP bleaching
1h 15m
Tissue sections were placed in a transparent box, which was then filled with the bleaching solution containing 2M H₂O₂ and 3mM EDTA in PBS at pH 12.5. The transparent box, holding the tissue slides and
bleaching solution, is positioned between two 5000 lux light pads (HSK, 615517997868) for one hour to facilitate fluorophore inactivation.
1h
TBST wash for 3 times, 5 minutes each
15m
Blocking
16h
Tissue sections were blocked with Blocker‱ casein (Thermo Scientific, 37528) in 4 C fridge overnight
16h
Staining
Normal IF, TSA, CODEX or IMC could be applied to the destaining slides after the blocking